Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

• 5.The international program conducted on the validation of parameters suitable to potentially detect endocrine activity of a test chemical showed that the quality of data obtained by this Test Method B.7 will depend much on the experience of the test laboratory. This relates specifically to the histopathological determination of cyclic changes in the female reproductive organs and to the weight determination of the small hormone dependent organs which are difficult to dissect. Guidance on histopathology has been developed (19). It is available on the OECD public website on Test Guidelines. It is intended to assist pathologists in their examinations and help increase the sensitivity of the assay. A variety of parameters were found to be indicative of endocrine-related toxicity and have been incorporated in the Test Method. Parameters for which insufficient data were available to prove usefulness or which showed only weak evidence in the validation programme of their ability to help in detection of endocrine disrupters are proposed as optional endpoints (see Appendix 2).

• 6.On the basis of data generated in the validation process, it must be emphasised that the sensitivity of this assay is not sufficient to identify all substances with (anti)androgenic or (anti)oestrogenic modes of action (9). The Test Method is not performed in a life-stage that is most sensitive to endocrine disruption. The Test Method nevertheless, during the validation process identified substances weakly and strongly affecting thyroid function, and strong and moderate endocrine active substances acting through oestrogen or androgen receptors, but in most cases failed to identify endocrine active substances that weakly affect oestrogen or androgen receptors. Thus it cannot be described as a screening assay for endocrine activity.

• 7.Consequently, the lack of effects related to these modes of action can not be taken as evidence for the lack of effects on the endocrine system. Regarding endocrine mediated effects, substance characterisation should not therefore be based on the results of this Test Method alone but should be used in a weight of evidence approach incorporating all existing data on a chemical to characterise potential endocrine activity. For this reason, regulatory decision making on endocrine activity (substance characterisation) should be a broadly based approach, not solely reliant on results from application of this test method.

• 8.It is acknowledged that all animal-based procedures will conform to local standards of animal care; the descriptions of care and treatment set forth below are minimal performance standards, and will be superseded by local regulations where more stringent. Further guidance of the humane treatment of animals is given by the OECD (14).

• 9.Definitions used are given in Appendix 1.

PRINCIPLE OF THE TEST

• 10.The test chemical is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 28 days. During the period of administration the animals are observed closely, each day for signs of toxicity. Animals that die or are euthanised during the test are necropsied and at the conclusion of the test surviving animals are euthanised and necropsied. A 28 day study provides information on the effects of repeated oral exposure and can indicate the need for further longer term studies. It can also provide information on the selection of concentrations for longer term studies. The data derived from using the Test Method should allow for the characterisation of the test chemical toxicity, for an indication of the dose response relationship and the determination of the No-Observed Adverse Effect Level (NOAEL).

DESCRIPTION OF THE METHOD

Selection of animal species

• 11.The preferred rodent species is the rat, although other rodent species may be used. If the parameters specified within this Test Method B.7 are investigated in another rodent species a detailed justification should be given. Although it is biologically plausible that other species should respond to toxicants in a similar manner to the rat, the use of smaller species may result in increased variability due to technical challenges of dissecting smaller organs. In the international validation program for the detection of endocrine disrupters, the rat was the only species used. Young healthy adult animals of commonly used laboratory strains should be employed. Females should be nulliparous and non pregnant. Dosing should begin as soon as feasible after weaning, and, in any case, before the animals are nine weeks old. At the commencement of the study the weight variation of animals used should be minimal and not exceed ± 20 % of the mean weight of each sex. When a repeated oral dose is conducted as a preliminary to a longer-term study, it is preferable that animals from the same strain and source should be used in both studies.

Housing and feeding

• 12.All procedures should conform to local standards of laboratory animal care. The temperature in the experimental animal room should be 22 °C (± 3 °C). Although the relative humidity should be at least 30 % and preferably not to exceed 70 % other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the photoperiod being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test chemical when administered by this method. Animals should be group housed in small groups of the same sex; animals may be housed individually if scientifically justified. For group caging, no more than five animals should be housed per cage.

• 13.The feed should be regularly analysed for contaminants. A sample of the diet should be retained until finalisation of the report.

Preparation of animals

• 14.Healthy young adult animals are randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals are identified uniquely and kept in their cages for at least five days prior to the start of the treatment study to allow for acclimatisation to the laboratory conditions.

Preparation of doses

• 15.The test chemical is administered by gavage or via the diet or drinking water. The method of oral administration is dependent on the purpose of the study, and the physical/chemical/toxico-kinetic properties of the test chemical.

• 16.Where necessary, the test chemical is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/suspension in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test chemical in the vehicle should be determined.

PROCEDURE

Number and sex of animals

• 17.At least 10 animals (five female and five male) should be used at each dose level. If interim euthanasia are planned, the number should be increased by the number of animals scheduled to be euthanised before the completion of the study. Consideration should be given to an additional satellite group of ten animals (five per sex) in the control and in the top dose group for observation of reversibility, persistence, or delayed occurrence of toxic effects, for at least 14 days post treatment.

Dosage

• 18.Generally, at least three test groups and a control group should be used, but if from assessment of other data, no effects would be expected at a dose of 1 000 mg/kg bw/d, a limit test may be performed. If there are no suitable data available, a range finding study (animals of the same strain and source) may be performed to aid the determination of the doses to be used. Except for treatment with the test chemical, animals in the control group should be handled in an identical manner to the test group subjects. If a vehicle is used in administering the test chemical, the control group should receive the vehicle in the highest volume used.

• 19.Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test chemical or related chemicals. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and no-observed-adverse effects at the lowest dose level (NOAEL). Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of 10) between dosages.

• 20.In the presence of observed general toxicity (e.g. reduced body weight, liver, heart, lung or kidney effects, etc.) or other changes that may not be toxic responses (e.g. reduced food intake, liver enlargement), observed effects on immune, neurological or endocrine sensitive endpoints should be interpreted with caution.

Limit test

• 21.If a test at one dose level of at least 1 000 mg/kg body weight/day or, for dietary or drinking water administration, an equivalent percentage in the diet, or drinking water (based upon body weight determinations), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data from structurally related chemicals, then a full study using three dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.

Administration of doses

• 22.The animals are dosed with test chemical daily 7 days each week for a period of 28 days. When the test chemical is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should not exceed 1 ml/100 g body weight except in the case of aqueous solutions where 2 ml/100 g body weight may be used. Except for irritating or corrosive chemicals, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimized by adjusting the concentration to ensure a constant volume at all dose levels.

• 23.For chemicals administered via the diet or drinking water it is important to ensure that the quantities of the test chemical involved do not interfere with normal nutrition or water balance. When the test chemical is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animals’ body weight may be used; the alternative used must be specified. For a chemical administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a repeated dose study is used as a preliminary to a long term study, a similar diet should be used in both studies.

Observations

• 24.The observation period should be 28 days. Animals in a satellite group scheduled for follow-up observations should be kept for at least 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

• 25.General clinical observations should be made at least once a day, preferably at the same time(s) each day and considering the peak period of anticipated effects after dosing. The health condition of the animals should be recorded. At least twice daily, all animals are observed for morbidity and mortality.

• 26.Once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time of day on each occasion. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) should also be recorded (2).

• 27.In the fourth exposure week sensory reactivity to stimuli of different types (2) (e.g. auditory, visual and proprioceptive stimuli) (3)(4)(5), assessment of grip strength (6) and motor activity assessment (7) should be conducted. Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could be used.

• 28.Functional observations conducted in the fourth exposure week may be omitted when the study is conducted as a preliminary study to a subsequent subchronic (90-day) study. In that case, the functional observations should be included in this follow-up study. On the other hand, the availability of data on functional observations from the repeated dose study may enhance the ability to select dose levels for a subsequent subchronic study.

• 29.As an exception, functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance.

• 30.At necropsy, the oestrus cycle of all females could be determined (optional) by taking vaginal smears. These observations will provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of estrogen sensitive tissues [see guidance on histopathology (19)].

Body weight and food/water consumption

• 31.All animals should be weighed at least once a week. Measurements of food consumption should be made at least weekly. If the test chemical is administered via the drinking water, water consumption should also be measured at least weekly.

Haematology

• 32.The following haematological examinations should be made at the end of the test period: haematocrit, haemoglobin concentrations, erythrocyte count, reticulocytes, total and differential leucocyte count, platelet count and a measure of blood clotting time/potential. Other determinations that should be carried out, if the test chemical or its putative metabolites have or are suspected to have oxidising properties include methaemoglobin concentration and Heinz bodies.

• 33.Blood samples should be taken from a named site just prior to or as part of the procedure for euthanasia of the animals, and stored under appropriate conditions. Animals should be fasted overnight prior to euthanasia ([^E0012]).

Clinical biochemistry

• 34.Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained of all animals just prior to or as part of the procedure for euthanasia of the animals (apart from those found moribund and/or euthanised prior to the termination of the study). Investigations of plasma or serum shall include sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanin aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl trans-peptidase and glutamate dehydrogenase), and bile acids. Measurements of additional enzymes (of hepatic or other origin) and bilirubin may provide useful information under certain circumstances.

• 35.Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.

• 36.In addition, studies to investigate plasma or serum markers of general tissue damage should be considered. Other determinations that should be carried out, if the known properties of the test chemical may, or are suspected to, affect related metabolic profiles include calcium, phosphate, triglycerides, specific hormones, and cholinesterase. These need to be identified for chemicals in certain classes or on a case-by-case basis.

• 37.Although in the international evaluation of the endocrine related endpoints a clear advantage for the determination of thyroid hormones (T3, T4) and TSH could not be demonstrated, it may be helpful to retain plasma or serum samples to measure T3, T4 and TSH (optional) if there is an indication for an effect on the pituitary-thyroid axis. These samples may be frozen at – 20° for storage. The following factors may influence the variability and the absolute concentrations of the hormone determinations:

• — time of sacrifice because of diurnal variation of hormone concentrations

• — method of sacrifice to avoid undue stress to the animals that may affect hormone concentrations

• — test kits for hormone determinations that may differ by their standard curves.

• Definitive identification of thyroid-active chemicals is more reliable by histopathological analysis rather than hormone levels.

• 38.Plasma samples specifically intended for hormone determination should be obtained at a comparable time of the day. It is recommended that consideration should be given to T3, T4 and TSH determinations triggered based upon alterations of thyroid histopathology. The numerical values obtained when analysing hormone concentrations differ with various commercial assay kits. Consequently, it may not be possible to provide performance criteria based upon uniform historical data. Alternatively, laboratories should strive to keep control coefficients of variation below 25 for T3 and T4 and below 35 for TSH. All concentrations are to be recorded in ng/ml.

• 39.If historical baseline data are inadequate, consideration should be given to determination of haematological and clinical biochemistry variables before dosing commences or preferably in a set of animals not included in the experimental groups.

PATHOLOGY

Gross necropsy

• 40.All animals in the study shall be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, epididymides, prostate + seminal vesicles with coagulating glands as a whole, thymus, spleen, brain and heart of all animals (apart from those found moribund and/or euthanised prior to the termination of the study) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying. Care must be exercised when trimming the prostate complex to avoid puncture of the fluid filled seminal vesicles. Alternatively, seminal vesicles and prostate may be trimmed and weighed after fixation.

• 41.In addition, two other tissues could be optionally weighed as soon as possible after dissection, to avoid drying: paired ovaries (wet weight) and uterus, including cervix (guidance on removal and preparation of the uterine tissues for weight measurement is provided in OECD TG 440 (18)).

• 42.The thyroid weight (optional) could be determined after fixation. Trimming should also be done very carefully and only after fixation to avoid tissue damage. Tissue damage could compromise histopathology analysis.

• 43.The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination (see paragraph 47): all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, eye, stomach, small and large intestines (including Peyer’s patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), gonads (testis and ovaries), accessory sex organs (uterus and cervix, epididymides, prostate + seminal vesicles with coagulating glands), vagina, urinary bladder, lymph nodes [besides the most proximal draining node another lymph node should be taken according to the laboratory’s experience (15)], peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, skeletal muscle and bone, with bone marrow (section or, alternatively, a fresh mounted bone marrow aspirate). It is recommended that testes be fixed by immersion in Bouin’s or modified Davidson’s fixative (16) (17). The tunica albuginea must be gently and shallowly punctured at the both poles of the organ with a needle to permit rapid penetration of the fixative. The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test chemical should be preserved.

• 44.The following tissues may give valuable indication for endocrine-related effects: Gonads (ovaries and testes), accessory sex organs (uterus including cervix, epididymides, seminal vesicles with coagulation glands, dorsolateral and ventral prostate), vagina, pituitary, male mammary gland, the thyroid and adrenal gland. Changes in male mammary glands have not been sufficiently documented but this parameter may be very sensitive to substances with oestrogenic action. Observation of organs/tissues that are not listed in paragraph 43 is optional (see Appendix 2).

• 45.The Guidance on histopathology (19) details extra information on dissection, fixation, sectioning and histopathology of endocrine tissues.

• 46.In the international test program some evidence was obtained that subtle endocrine effects by chemicals with a low potency for affecting sex hormone homeostasis may be identified by disturbance of the synchronisation of the oestrus cycle in different tissues and not so much by frank histopathological alterations in female sex organs. Although no definitive proof was obtained for such effects, it is recommended that evidence of possible asynchrony of the oestrus cycle should be taken into account in interpretation of the histopathology of the ovaries (follicular, thecal, and granulosa cells), uterus, cervix and vagina. If assessed, the stage of cycle as determined by vaginal smears could be included in this comparison as well.

Histopathology

• 47.Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups. These examinations should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group.

• 48.All gross lesions shall be examined.

• 49.When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.

DATA AND REPORTING

Data

• 50.Individual data should be provided. Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or euthanised for humane reasons and the time of any death or euthanasia, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions, their severity and the percentage of animals displaying each type of lesion.

• 51.When possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method. Comparisons of the effect along a dose range should avoid the use of multiple t-tests. The statistical methods should be selected during the design of the study.

• 52.For quality control it is proposed that historical control data are collected and that for numerical data coefficients of variation are calculated, especially for the parameters linked with endocrine disrupter detection. These data can be used for comparison purposes when actual studies are evaluated.

Test report

• 53.The test report must include the following information:

Test chemical:

• — physical nature, purity and physicochemical properties;

• — identification data.

Vehicle (if appropriate):

• — justification for choice of vehicle, if other than water.

Test animals:

• — species/strain used;

• — number, age and sex of animals;

• — source, housing conditions, diet, etc.;

• — individual weights of animals at the start of the test.

• — justification for species if not rat

Test conditions:

• — rationale for dose level selection;

• — details of test chemical formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation;

• — details of the administration of the test chemical;

• — conversion from diet/drinking water test chemical concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable;

• — details of food and water quality.

Optional endpoints investigated

• — list of optional endpoints investigated

Results:

• — body weight/body weight changes;

• — food consumption, and water consumption, if applicable;

• — toxic response data by sex and dose level, including signs of toxicity;

• — nature, severity and duration of clinical observations (whether reversible or not);

• — sensory activity, grip strength and motor activity assessments;

• — haematological tests with relevant base-line values;

• — clinical biochemistry tests with relevant base-line values;

• — body weight at euthanasia and organ weight data;

• — necropsy findings;

• — a detailed description of all histopathological findings;

• — absorption data if available;

• — statistical treatment of results, where appropriate.

Discussion of results

Conclusions

Appendix 1

DEFINITIONS

Androgenicity is the capability of a chemical to act like a natural androgenic hormone (e.g. testosterone) in a mammalian organism.

Antiandrogenicity is the capability of a chemical to suppress the action of a natural androgenic hormone (e.g. testosterone) in a mammalian organism.

Antioestrogenicity is the capability of a chemical to suppress the action of a natural oestrogenic hormone (e.g. oestradiol 17ß) in a mammalian organism.

Antithyroid activity is the capability of a chemical to suppress the action of a natural thyroid hormone (e.g. T3) in a mammalian organism.

Dosage is a general term comprising of dose, its frequency and the duration of dosing.

Dose is the amount of test chemical administered. The dose is expressed as weight of test chemical per unit body weight of test animal per day (e.g. mg/kg body weight/day), or as a constant dietary concentration.

Evident toxicity is a general term describing clear signs of toxicity following administration of test chemical. These should be sufficient for hazard assessment and should be such that an increase in the dose administered can be expected to result in the development of severe toxic signs and probable mortality.

NOAEL is the abbreviation for no-observed-adverse-effect level. This is the highest dose level where no adverse treatment-related findings are observed due to treatment.

Oestrogenicity is the capability of a chemical to act like a natural oestrogenic hormone (e.g. oestradiol 17ß) in a mammalian organism.

Test chemical: Any substance or mixture tested using this Test Method.

Thyroid activity is the capability of a chemical to act like a natural thyroid hormone (e.g. T3) in a mammalian organism.

Validation is a scientific process designed to characterise the operational requirements and limitations of a test method and to demonstrate its reliability and relevance for a particular purpose.

Appendix 2

Endpoints recommended for the detection of endocrine disrupters (EDs) in this Test Method B.7

Mandatory endpoints	Optional endpoints
Weight	Weight
— Testes — Epididymides — Adrenals — Prostate + seminal vesicles with coagulating glands	— Ovaries — Uterus, including cervix — Thyroid
Histopathology	Histopathology
— Gonads: — — Testes and — Ovaries — Accessory sex organs: — — Epididymides, — Prostate + seminal vesicle with coagulating glands — Uterus, including cervix — Adrenal — Thyroid — Vagina	— Vaginal smears — Male mammary glands — Pituitary
Hormones measurement	Hormones measurement
	— Circulating levels of T3, T4 — Circulating levels of TSH

LITERATURE:

• (1) OECD (Paris, 1992). Chairman’s Report of the Meeting of the ad hoc Working Group of Experts on Systemic Short-term and (Delayed) Neurotoxicity.

• (2) IPCS (1986). Principles and Methods for the Assessment of Neurotoxicity Associated with Exposure to Chemicals. Environmental Health Criteria Document No 60.

• (3) Tupper DE, Wallace RB (1980). Utility of the Neurologic Examination in Rats. Acta Neurobiol. Exp. 40: 999-1003.

• (4) Gad SC (1982). A Neuromuscular Screen for Use in Industrial Toxicology. J. Toxicol Environ. Health 9: 691-704.

• (5) Moser VC, McDaniel KM, Phillips PM (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108: 267-283.

• (6) Meyer OA, Tilson HA, Byrd WC, Riley MT (1979). A Method for the Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice. Neurobehav. Toxicol. 1: 233-236.

• (7) Crofton KM, Howard JL, Moser VC, Gill MW, Reiter LW, Tilson HA, MacPhail RC (1991). Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol. 13: 599-609.

• (8) OECD (1998). Report of the First Meeting of the OECD Endocrine Disrupter Testing and Assessment (EDTA) Task Force, 10th-11 March 1998, ENV/MC/CHEM/RA(98)5.

• (9) OECD. (2006). Report of the Validation of the Updated Test Guideline 407: Repeat Dose 28-day Oral Toxicity Study in Laboratory Rats. Series on Testing and Assessment No 59, ENV/JM/MONO(2006)26.

• (10) OECD (2002). Detailed Review Paper on the Appraisal of Test Methods for Sex Hormone Disrupting Chemicals. Series on Testing and Assessment No 21, ENV/JM/MONO(2002)8.

• (11) OECD (2012).Conceptual Framework for Testing and Assessment of Endocrine Disrupting Chemicals. http://www.oecd.org/document/58/0,3343,fr_2649_37407_2348794_1_1_1_37407,00.html

• (12) OECD (2006). Final Summary report of the meeting of the Validation Management Group for mammalian testing. ENV/JM/TG/EDTA/M(2006)2.

• (13) OECD. Draft Summary record of the meeting of the Task Force on Endocrine Disrupters Testing and Assessment. ENV/JM/TG/EDTA/M(2006)3.

• (14) OECD (2000). Guidance document on the recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation. Series on Testing and Assessment No 19. ENV/JM/MONO(2000)7.

• (15) Haley P, Perry R, Ennulat D, Frame S, Johnson C, Lapointe J-M, Nyska A, Snyder PW, Walker D, Walter G (2005). STP Position Paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System. Toxicol Pathol 33: 404-407.

• (16) Hess RA, Moore BJ (1993). Histological Methods for the Evaluation of the Testis. In: Methods in Reproductive Toxicology, Chapin RE and Heindel JJ (eds). Academic Press: San Diego, CA, pp. 52-85.

• (17) Latendresse JR, Warbrittion AR, Jonassen H, Creasy DM.(2002) Fixation of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicol. Pathol. 30, 524-533.

• (18) OECD (2007). OECD Guideline for Testing of Chemicals No 440: Uterotrophic Bioassay in Rodents: A short-term screening test for oestrogenic properties.

• (19) OECD (2009). Guidance Document 106 on Histologic evaluation of Endocrine and Reproductive Tests in Rodents ENV/JM/Mono(2009)11.

B.8.   SUBACUTE INHALATION TOXICITY: 28-DAY STUDY

The full description of this test method has been deleted. The equivalent international test method appears in Table 2 of Part 0.

B.9.   REPEATED DOSE (28 DAYS) TOXICITY (DERMAL)

1. METHOD

1.1.   INTRODUCTION

See General introduction Part B (A).

1.2.   DEFINITIONS

See General introduction Part B (B).

1.3.   REFERENCE SUBSTANCES

None.

1.4.   PRINCIPLE OF THE TEST METHOD

The test substance is applied daily to the skin in graduated doses to several groups of experimental animals, one dose per group, for a period of 28 days. During the period of application, the animals are observed daily to detect signs of toxicity. Animals, which die during the test, are necropsied and at the conclusion of the test surviving animals are necropsied.

1.5.   QUALITY CRITERIA

None.

1.6.   DESCRIPTION OF THE TEST METHOD

1.6.1.   Preparations

The animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. Before the test, healthy young animals are randomised and assigned to the treatment and control groups. Shortly before testing, fur is clipped from the dorsal area of the trunk of the test animals. Shaving may be employed but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care must be taken to avoid abrading the skin. Not less than 10 % of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of the covering. When testing solids, which may be pulverised if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. Liquid test substances are generally used undiluted. Daily application on a five to seven-day per week basis is used.

1.6.2.   Test conditions

1.6.2.1.   Experimental animals

The adult rat, rabbit or guinea-pig may be used. Other species may be used but their use would require justification.

At the commencement of the study, the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.

1.6.2.2.   Number and sex

At least 10 animals (five female and five male) with healthy skin should be used at each dose level. The females should be nulliparous and non-pregnant. If interim sacrifices are planned, the numbers should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 10 animals (five animals per sex) may be treated with the high dose level for 28 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 14 days post-treatment. A satellite group of 10 control animals (five animals per sex) is also used.

1.6.2.3.   Dose levels

At least three dose levels are required with a control or a vehicle control if a vehicle is used. The exposure period should be at least six hours per day. The application of the test substance should be made at similar times each day, and adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body-weight. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. Where a vehicle is used to facilitate dosing, the vehicle control group should be dosed in the same way as the treated groups, and receive the same amount as that received by the highest dose level group. The highest dose level should result in toxic effects but produce no, or few, fatalities. The lowest dose level should not produce any evidence or toxicity. Where there is a usable estimation of human exposure, the lowest level should exceed this. Ideally, the intermediate dose level should produce minimal observable toxic effects. If more than one intermediate dose is used the dose levels should be spaced to produce a gradation of toxic effects. In the low and intermediate groups and in the controls, the incidence of fatalities should be low in order to permit a meaningful evaluation of the results.

If application of the test substance produces severe skin irritation, the concentrations should be reduced and this may result in a reduction in, or absence of, other toxic effects at the high dose level. Moreover if the skin has been badly damaged it may be necessary to terminate the study and undertake a new study at lower concentrations.

1.6.2.4.   Limit test

If a preliminary study at a dose level of 1 000 mg/kg, or a higher dose level related to possible human exposure where this is known, produces no toxic effects, further testing may not be considered necessary.

1.6.2.5.   Observation period

The experimental animals should be observed daily for signs of toxicity. The time of death and the time at which signs of toxicity appear and disappear should be recorded.

1.6.3.   Procedure

Animals should be caged individually. The animals are treated with the test substance, ideally on seven days per week, for a period of 28 days. Animals in any satellite groups scheduled for follow-up observations should be kept for a further 14 days without treatment to detect recovery from or persistence of toxic effects. Exposure time should be at least six hours per day.

The test substance should be applied uniformly over an area, which is approximately 10 % of the total body surface area. With highly toxic substances, the surface area covered may be less but as much of the area as possible should be covered with as thin and uniform a layer as possible.

During exposure the test substance is held in contact with the skin with porous gauze dressing and non-irritating tape. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance but complete immobilisation is not a recommended method. As an alternative a ‘collar protective device’ may be used.

At the end of the exposure period, residual test substance should be removed, where practicable, using water or some other appropriate method of cleansing the skin.

All the animals should be observed daily and signs of toxicity recorded including the time of onset, their degree and duration. Observations should include changes in skin and fur, eyes and mucous membranes as well as respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Measurements should be made weekly of the animals' weight. It is also recommended that food consumption is measured weekly. Regular observation of the animals is necessary to ensure that animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the study period, all survivors in the non-satellite treatment groups are necropsied. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.

The following examinations shall be made at the end of the test on all animals including the controls:

• (1) haematology, including at least haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count, and a measure of clotting potential;

• (2) clinical blood biochemistry including at least one parameter of liver and kidney function: serum alanine aminotransferase (formerly known as glutamic pyruvic transaminase), serum aspartate aminotransferase (formerly known as glutamic oxaloacetic transaminase), urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein;

Other determinations which may be necessary for an adequate toxicological evaluation include calcium, phosphorus, chloride, sodium, potassium, fasting glucose, analysis of lipids, hormones, acid/base balance, methaemoglobin and cholinesterase activity.

Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.

1.6.4.   Gross necropsy

All animals in the study should be subjected to a full gross necropsy. At least the liver, kidneys, adrenals, and testes should be weighed wet as soon as possible after dissection, to avoid drying. Organs and tissues, i.e. normal and treated skin, liver, kidney, spleen, testes, adrenals, heart, and target organs (that is those organs showing gross lesions or changes in size) should be preserved in a suitable medium for possible future histopathological examination.

1.6.5.   Histopathological examination

In the high dose group and in the control group, histological examination should be performed on the preserved organs and tissues. Organs and tissues showing defects attributable to the test substance at the highest dosage level should be examined in all lower-dosage groups. Animals in the satellite group should be examined histologically with particular emphasis on those organs and tissues identified as showing effects in the other treated groups.

2. DATA

Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test and the number of animals displaying each type of lesion.

All observed results should be evaluated by an appropriate statistical method. Any recognised statistical method may be used.

3. REPORTING

3.1.   TEST REPORT

The test report shall, if possible, include the following information:

• — animal data (species, strain, source, environmental conditions, diet, etc.),

• — test conditions (including the type of dressing: occlusive or not-occlusive),

• — dose levels (including vehicle, if used) and concentrations,

• — no-effect level, where possible,

• — toxic response data by sex and dose,

• — time of death during the study or whether animals survived to termination,

• — toxic or other effects,

• — the time of observation of each abnormal sign and its subsequent course,

• — food and body-weight data,

• — haematological tests employed and results,

• — clinical biochemistry tests employed and results,

• — necropsy findings,

• — a detailed description of all histopathological findings,

• — statistical treatment of results where possible,

• — discussion of the results,

• — interpretation of the results.

3.2.   EVALUATION AND INTERPRETATION

See General introduction Part B (D).

4. REFERENCES

See General introduction Part B (E).

B.10.   IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST

INTRODUCTION

This test method is equivalent to OECD test guideline 473 (2016). It is part of a series of test methods on genetic toxicology. An OECD document that provides succinct information on genetic toxicology testing and an overview of the recent changes that were made to these Test Guidelines has been developed (1).

The purpose of the in vitro chromosomal aberration test is to identify chemicals that cause structural chromosomal aberrations in cultured mammalian cells (2) (3) (4). Structural aberrations may be of two types, chromosome or chromatid. Polyploidy (including endoreduplication) could arise in chromosome aberration assays in vitro. While aneugens can induce polyploidy, polyploidy alone does not indicate aneugenic potential and can simply indicate cell cycle perturbation or cytotoxicity (5). This test is not designed to measure aneuploidy. An in vitro micronucleus test (6) would be recommended for the detection of aneuploidy.

The in vitro chromosomal aberration test may employ cultures of established cell lines or primary cell cultures of human or rodent origin. The cells used should be selected on the basis of growth ability in culture, stability of the karyotype (including chromosome number) and spontaneous frequency of chromosomal aberrations (7). At the present time, the available data do not allow firm recommendations to be made but suggest it is important, when evaluating chemical hazards to consider the p53 status, genetic (karyotype) stability, DNA repair capacity and origin (rodent versus human) of the cells chosen for testing. The users of this test method are thus encouraged to consider the influence of these and other cell characteristics on the performance of a cell line in detecting the induction of chromosomal aberrations, as knowledge evolves in this area.

Definitions used are provided in Appendix 1.

INITIAL CONSIDERATIONS AND LIMITATIONS

Tests conducted in vitro generally require the use of an exogenous source of metabolic activation unless the cells are metabolically competent with respect to the test chemicals. The exogenous metabolic activation system does not entirely mimic in vivo conditions. Care should be taken to avoid conditions that could lead to artifactual positive results, i.e. chromosome damage not caused by direct interaction between the test chemicals and chromosomes; such conditions include changes in pH or osmolality (8) (9) (10), interaction with the medium components (11) (12) or excessive levels of cytotoxicity (13) (14) (15) (16).

This test is used to detect chromosomal aberrations that may result from clastogenic events. The analysis of chromosomal aberration induction should be done using cells in metaphase. It is thus essential that cells should reach mitosis both in treated and in untreated cultures. For manufactured nanomaterials, specific adaptations of this test method may be needed but are not described in this test method.

Before use of the test method on a mixture for generating data for an intended regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose. Such considerations are not needed, when there is a regulatory requirement for testing of the mixture.

PRINCIPLE OF THE TEST

Cell cultures of human or other mammalian origin are exposed to the test chemical both with and without an exogenous source of metabolic activation unless cells with an adequate metabolizing capability are used (see paragraph 13). At appropriate predetermined intervals after the start of exposure of cell cultures to the test chemical, they are treated with a metaphase-arresting chemical (e.g. colcemid or colchicine), harvested, stained and metaphase cells are analysed microscopically for the presence of chromatid-type and chromosome-type aberrations.

DESCRIPTION OF THE METHOD

Preparations

Cells

A variety of cell lines (e.g. Chinese Hamster Ovary (CHO), Chinese Hamster lung V79, Chinese Hamster Lung (CHL)/IU, TK6) or primary cell cultures, including human or other mammalian peripheral blood lymphocytes, can be used (7). The choice of the cell lines used should be scientifically justified. When primary cells are used, for animal welfare reasons, the use of primary cells from human origin should be considered where feasible and sampled in accordance with the human ethical principles and regulations. Human peripheral blood lymphocytes should be obtained from young (approximately 18-35 years of age), non-smoking individuals with no known illness or recent exposures to genotoxic agents (e.g. chemicals, ionizing radiations) at levels that would increase the background incidence of chromosomal aberrations. This would ensure the background incidence of chromosomal aberrations to be low and consistent. The baseline incidence of chromosomal aberrations increases with age and this trend is more marked in females than in males (17) (18). If cells from more than one donor are pooled for use, the number of donors should be specified. It is necessary to demonstrate that the cells have divided from the beginning of treatment with the test chemical to cell sampling. Cell cultures are maintained in an exponential cell growth phase (cell lines) or stimulated to divide (primary cultures of lymphocytes), to expose the cells at different stages of the cell cycle, since the sensitivity of cell stages to the test chemicals may not be known. The primary cells that need to be stimulated with mitogenic agents in order to divide are generally no longer synchronized during exposure to the test chemical (e.g. human lymphocytes after a 48-hour mitogenic stimulation). The use of synchronized cells during treatment is not recommended, but can be acceptable if justified.

Media and culture conditions

Appropriate culture medium and incubation conditions (culture vessels, humidified atmosphere of 5 % CO2 if appropriate, incubation temperature of 37 °C) should be used for maintaining cultures. Cell lines should be checked routinely for the stability of the modal chromosome number and the absence of Mycoplasma contamination (7) (19), and cells should not be used if contaminated or if the modal chromosome number has changed. The normal cell cycle time of cell lines or primary cultures used in the testing laboratory should be established and should be consistent with the published cell characteristics (20).

Preparation of cultures

Cell lines: cells are propagated from stock cultures, seeded in culture medium at a density such that the cells in suspensions or in monolayers will continue to grow exponentially until harvest time (e.g. confluence should be avoided for cells growing in monolayers).

Lymphocytes: whole blood treated with an anti-coagulant (e.g. heparin) or separated lymphocytes are cultured (e.g. for 48 hours for human lymphocytes) in the presence of a mitogen [e.g. phytohaemagglutinin (PHA) for human lymphocytes] in order to induce cell division prior to exposure to the test chemical.

Metabolic activation

Exogenous metabolising systems should be used when employing cells which have inadequate endogenous metabolic capacity. The most commonly used system that is recommended by default, unless otherwise justified, is a co-factor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents (generally rats) treated with enzyme-inducing agents such as Aroclor 1254 (21) (22) (23) or a combination of phenobarbital and β-naphthoflavone (24) (25) (26) (27) (28) (29). The latter combination does not conflict with the Stockholm Convention on Persistent Organic Pollutants (30) and has been shown to be as effective as Aroclor 1254 for inducing mixed-function oxidases (24) (25) (26) (28). The S9 fraction typically is used at concentrations ranging from 1 to 2 % (v/v) but may be increased to 10 % (v/v) in the final test medium. The use of products that reduce the mitotic index, especially calcium complexing products (31) should be avoided during treatment. The choice of type and concentration of exogenous metabolic activation system or metabolic inducer employed may be influenced by the class of chemicals being tested.

Test chemical preparation

Solid test chemicals should be prepared in appropriate solvents and diluted, if appropriate, prior to treatment of the cells (see paragraph 23). Liquid test chemicals may be added directly to the test system and/or diluted prior to treatment of the test system. Gaseous or volatile test chemicals should be tested by appropriate modifications to the standard protocols, such as treatment in sealed culture vessels (32) (33) (34). Preparations of the test chemical should be made just prior to treatment unless stability data demonstrate the acceptability of storage.

Test conditions

Solvents

The solvent should be chosen to optimize the solubility of the test chemicals without adversely impacting the conduct of the assay, e.g. changing cell growth, affecting the integrity of the test chemical, reacting with culture vessels, impairing the metabolic activation system. It is recommended that, wherever possible, the use of an aqueous solvent (or culture medium) should be considered first. Well established solvents are for example water or dimethyl sulfoxide. Generally organic solvents should not exceed 1 % (v/v) and aqueous solvents (saline or water) should not exceed 10 % (v/v) in the final treatment medium. If not well-established solvents are used (e.g. ethanol or acetone), their use should be supported by data indicating their compatibility with the test chemicals, the test system and their lack of genetic toxicity at the concentration used. In the absence of that supporting data, it is important to include untreated controls (see Appendix 1) to demonstrate that no deleterious or clastogenic effects are induced by the chosen solvent.

Measuring cell proliferation and cytotoxicity and choosing treatment concentrations

When determining the highest test chemical concentration, concentrations that have the capability of producing artifactual positive responses, such as those producing excessive cytotoxicity (see paragraph 22), precipitation in the culture medium (see paragraph 23), or marked changes in pH or osmolality (see paragraph 5), should be avoided. If the test chemical causes a marked change in the pH of the medium at the time of addition, the pH might be adjusted by buffering the final treatment medium so as to avoid artifactual positive results and to maintain appropriate culture conditions.

Measurements of cell proliferation are made to assure that a sufficient number of treated cells have reached mitosis during the test and that the treatments are conducted at appropriate levels of cytotoxicity (see paragraphs 18 and 22). Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indication of cell death and growth. While the evaluation of cytotoxicity in an initial test may be useful to better define the concentrations to be used in the main experiment, an initial test is not mandatory. If performed, it should not replace the measurement of cytotoxicity in the main experiment.

Relative Population Doubling (RPD) or Relative Increase in Cell Count (RICC) are appropriate methods for the assessment of cytotoxicity in cytogenetic tests (13) (15) (35) (36) (55) (see Appendix 2 for formulas). In case of long-term treatment and sampling times after the beginning of treatment longer than 1,5 normal cell cycle lengths (i.e. longer than 3 cell cycle lengths in total), RPD might underestimate cytotoxicity (37). Under these circumstances RICC might be a better measure or the evaluation of cytotoxicity after 1,5 normal cell cycle lengths would be a helpful estimate using RPD.

For lymphocytes in primary cultures, while the mitotic index (MI) is a measure of cytotoxic/cytostatic effects, it is influenced by the time after treatment it is measured, the mitogen used and possible cell cycle disruption. However, the MI is acceptable because other cytotoxicity measurements may be cumbersome and impractical and may not apply to the target population of lymphocytes growing in response to PHA stimulation.

While RICC and RPD for cell lines and MI for primary culture of lymphocytes are the recommended cytotoxicity parameters, other indicators (e.g. cell integrity, apoptosis, necrosis, cell cycle) could provide useful additional information.

At least three test concentrations (not including the solvent and positive controls) that meet the acceptability criteria (appropriate cytotoxicity, number of cells, etc) should be evaluated. Whatever the types of cells (cell lines or primary cultures of lymphocytes), either replicate or single treated cultures may be used at each concentration tested. While the use of duplicate cultures is advisable, single cultures are also acceptable provided that the same total number of cells are scored for either single or duplicate cultures. The use of single cultures is particularly relevant when more than 3 concentrations are assessed (see paragraph 31). The results obtained in the independent replicate cultures at a given concentration can be pooled for the data analysis (38). For test chemicals demonstrating little or no cytotoxicity, concentration intervals of approximately 2 to 3 fold will usually be appropriate. Where cytotoxicity occurs, the test concentrations selected should cover a range from that producing cytotoxicity as described in paragraph 22 and including concentrations at which there is moderate and little or no cytotoxicity. Many test chemicals exhibit steep concentration response curves and in order to obtain data at low and moderate cytotoxicity or to study the dose response relationship in detail, it will be necessary to use more closely spaced concentrations and/or more than three concentrations (single cultures or replicates), in particular in situations where a repeat experiment is required (see paragraph 47).

If the maximum concentration is based on cytotoxicity, the highest concentration should aim to achieve 55 ± 5 % cytotoxicity using the recommended cytotoxicity parameters (i.e. reduction in RICC and RPD for cell lines and reduction in MI for primary cultures of lymphocytes to 45 ± 5 % of the concurrent negative control). Care should be taken in interpreting positive results only to be found in the higher end of this 55 ± 5 % cytotoxicity range (13).

For poorly soluble test chemicals that are not cytotoxic at concentrations lower than the lowest insoluble concentration, the highest concentration analysed should produce turbidity or a precipitate visible by eye or with the aid of an inverted microscope at the end of the treatment with the test chemical. Even if cytotoxicity occurs above the lowest insoluble concentration, it is advisable to test at only one concentration producing turbidity or with a visible precipitate because artifactual effects may result from the precipitate. At the concentration producing a precipitate, care should be taken to assure that the precipitate does not interfere with the conduct of the test (e.g. staining or scoring). The determination of solubility in the culture medium prior to the experiment may be useful.

If no precipitate or limiting cytotoxicity is observed, the highest test concentration should correspond to 10 mM, 2 mg/ml or 2 μl/ml, whichever is the lowest (39) (40) (41). When the test chemical is not of defined composition, e.g. a substance of unknown or variable composition, complex reaction products or biological material (UVCB) (42), environmental extract etc., the top concentration may need to be higher (e.g. 5 mg/ml), in the absence of sufficient cytotoxicity, to increase the concentration of each of the components. It should be noted however that these requirements may differ for human pharmaceuticals (43).

Controls

Concurrent negative controls (see paragraph 15), consisting of solvent alone in the treatment medium and treated in the same way as the treatment cultures, should be included for every harvest time.

Concurrent positive controls are needed to demonstrate the ability of the laboratory to identify clastogens under the conditions of the test protocol used and the effectiveness of the exogenous metabolic activation system, when applicable. Examples of positive controls are given in the table 1 below. Alternative positive control chemicals can be used, if justified. Because in vitro mammalian cell tests for genetic toxicity are sufficiently standardized, the use of positive controls may be confined to a clastogen requiring metabolic activation. Provided it is done concurrently with the non-activated test using the same treatment duration, this single positive control response will demonstrate both the activity of the metabolic activation system and the responsiveness of the test system. Long term treatment (without S9) should however have its own positive control as the treatment duration will differ from the test using metabolic activation. Each positive control should be used at one or more concentrations expected to give reproducible and detectable increases over background in order to demonstrate the sensitivity of the test system (i.e. the effects are clear but do not immediately reveal the identity of the coded slides to the reader), and the response should not be compromised by cytotoxicity exceeding the limits specified in the test method.

Table 1

Reference chemicals recommended for assessing laboratory proficiency and for selection of positive controls

Category	Chemical	CASRN
1. Clastogens active without metabolic activation	1. Clastogens active without metabolic activation	1. Clastogens active without metabolic activation
	Methyl methanesulphonate	66-27-3
	Mitomycin C	50-07-7
	4-Nitroquinoline-N-Oxide	56-57-5
	Cytosine arabinoside	147-94-4
2. Clastogens requiring metabolic activation	2. Clastogens requiring metabolic activation	2. Clastogens requiring metabolic activation
	Benzo(a)pyrene	50-32-8
	Cyclophosphamide	50-18-0

PROCEDURE

Treatment with test chemical

Proliferating cells are treated with the test chemical in the presence and absence of a metabolic activation system.

Culture harvest time

For thorough evaluation, which would be needed to conclude a negative outcome, all three of the following experimental conditions should be conducted using a short term treatment with and without metabolic activation and long term treatment without metabolic activation (see paragraphs 43, 44 and 45):

• — Cells should be exposed to the test chemical without metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1,5 normal cell cycle lengths after the beginning of treatment (18),

• — Cells should be exposed to the test chemical with metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1,5 normal cell cycle lengths after the beginning of treatment (18),

• — Cells should be continuously exposed without metabolic activation until sampling at a time equivalent to about 1,5 normal cell cycle lengths. Certain chemicals (e.g. nucleoside analogues) may be more readily detected by treatment/sampling times longer than 1,5 normal cell cycle lengths (24).

In the event that any of the above experimental conditions lead to a positive response, it may not be necessary to investigate any of the other treatment regimens.

Chromosome preparation

Cell cultures are treated with colcemid or colchicine usually for one to three hours prior to harvesting. Each cell culture is harvested and processed separately for the preparation of chromosomes. Chromosome preparation involves hypotonic treatment of the cells, fixation and staining. In monolayers, mitotic cells (identifiable as being round and detaching from the surface) may be present at the end of the 3-6 hour treatment. Because these mitotic cells are easily detached, they can be lost when the medium containing the test chemical is removed. If there is evidence for a substantial increase in the number of mitotic cells compared with controls, indicating likely mitotic arrest, then the cells should be collected by centrifugation and added back to cultures, to avoid losing cells that are in mitosis, and at risk for chromosome aberration, at the time of harvest.

Analysis

All slides, including those of the positive and negative controls, should be independently coded before microscopic analysis for chromosomal aberrations. Since fixation procedures often result in a proportion of metaphase cells which have lost chromosomes, the cells scored should, therefore, contain a number of centromeres equal to the modal number +/- 2.

At least 300 well-spread metaphases should be scored per concentration and control to conclude a test chemical as clearly negative (see paragraph 45). The 300 cells should be equally divided among the replicates, when replicate cultures are used. When single cultures are used per concentration (see paragraph 21), at least 300 well spread metaphases should be scored in this single culture. Scoring 300 cells has the advantage of increasing the statistical power of the test and in addition, zero values will be rarely observed (expected to be only 5 %) (44). The number of metaphases scored can be reduced when high numbers of cells with chromosome aberrations are observed and the test chemical considered as clearly positive.

Cells with structural chromosomal aberration(s) including and excluding gaps should be scored. Breaks and gaps are defined in Appendix 1 according to (45) (46). Chromatid- and chromosome-type aberrations should be recorded separately and classified by sub-types (breaks, exchanges). Procedures in use in the laboratory should ensure that analysis of chromosomal aberrations is performed by well-trained scorers and peer-reviewed if appropriate.

Although the purpose of the test is to detect structural chromosomal aberrations, it is important to record polyploidy and endoreduplication frequencies when these events are seen. (See paragraph 2).

Proficiency of the laboratory

In order to establish sufficient experience with the test prior to using it for routine testing, the laboratory should have performed a series of experiments with reference positive chemicals acting via different mechanisms and various negative controls (using various solvents/vehicle). These positive and negative control responses should be consistent with the literature. This is not applicable to laboratories that have experience, i.e. that have an historical data base available as defined in paragraph 37.

A selection of positive control chemicals (see Table 1 in paragraph 26) should be investigated with short and long treatments in the absence of metabolic activation, and also with short treatment in the presence of metabolic activation, in order to demonstrate proficiency to detect clastogenic chemicals and determine the effectiveness of the metabolic activation system. A range of concentrations of the selected chemicals should be chosen so as to give reproducible and concentration-related increases above the background in order to demonstrate the sensitivity and dynamic range of the test system.

Historical control data

The laboratory should establish:

• — A historical positive control range and distribution,

• — A historical negative (untreated, solvent) control range and distribution.

When first acquiring data for an historical negative control distribution, concurrent negative controls should be consistent with published control data, where they exist. As more experimental data are added to the control distribution, concurrent negative controls should ideally be within the 95 % control limits of that distribution (44) (47). The laboratory's historical negative control database should initially be built with a minimum of 10 experiments but would preferably consist of at least 20 experiments conducted under comparable experimental conditions. Laboratories should use quality control methods, such as control charts (e.g. C-charts or X-bar charts (48)), to identify how variable their positive and negative control data are, and to show that the methodology is ‘under control’ in their laboratory (44). Further recommendations on how to build and use the historical data (i.e. criteria for inclusion and exclusion of data in historical data and the acceptability criteria for a given experiment) can be found in the literature (47).

Any changes to the experimental protocol should be considered in terms of their consistency with the laboratory's existing historical control databases. Any major inconsistencies should result in the establishment of a new historical control database.

Negative control data should consist of the incidence of cells with chromosome aberrations from a single culture or the sum of replicate cultures as described in paragraph 21. Concurrent negative controls should ideally be within the 95 % control limits of the distribution of the laboratory's historical negative control database (44) (47). Where concurrent negative control data fall outside the 95 % control limits they may be acceptable for inclusion in the historical control distribution as long as these data are not extreme outliers and there is evidence that the test system is ‘under control’ (see paragraph 37) and evidence of absence of technical or human failure.

DATA AND REPORTING

Presentation of the results

The percentage of cells with structural chromosomal aberration(s) should be evaluated. Chromatid- and chromosome-type aberrations classified by sub-types (breaks, exchanges) should be listed separately with their numbers and frequencies for experimental and control cultures. Gaps are recorded and reported separately but not included in the total aberration frequency. Percentage of polyploidy and/or endoreduplicated cells are reported when seen.

Concurrent measures of cytotoxicity for all treated, negative and positive control cultures in the main aberration experiment(s) should be recorded.

Individual culture data should be provided. Additionally, all data should be summarised in tabular form.

Acceptability Criteria

Acceptance of a test is based on the following criteria:

• — The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database as described in paragraph 39.